Regulating biocondensates within synthetic cells via segregative phase separation
Living cells orchestrate a myriad of biological reactions within a highly complex and crowded environment. A major factor responsible for such seamless assembly are the preferential interactions between the constituent macromolecules, either associative or segregative, that can drive de-mixing to produce co-existing phases, and thus provide a dynamic intracellular compartmentalization. But how these two types of interactions, occurring simultaneously within the cytoplasmic space, influence each other is still largely unknown. This makes understanding and applying the molecular interactions that interfere with each other in such crowded environments crucial when engineering increasingly complex synthetic cells. Here, we show that the interplay between segregative and associative phase separation within cell-mimicking vesicles can lead to rich dynamics between them. Using on-chip microfluidic systems, we encapsulate the associative and segregative components in cell-sized containers and trigger their phase separations to create hierarchical structures that act as molecular recruiters, membrane targeting agents, and initiators of condensation. The obtained multiphase architecture provides an isolated microenvironment for condensates, restricting their molecular communication as well as diffusive motion, and leading to budding-like behaviour at the lipid membrane. In conclusion, we propose segregative phase separation as a universal condensate regulation strategy in managing molecular distribution, condensate location, as well as membrane interaction. We believe our approach will facilitate controlling the behaviour of membraneless organelles within synthetic cells.